Several anion transport systems of the inner mitrochondrial membrane appear to function as proton compensated electroneutral transporters. It is the objective of this research proposal to identify the proteins of the inner mitochondrial membrane from rat brain and liver, beef heart, and blowfly flight muscle from developing and senescent animals that are responsible for pyruvate transport and glutamate transport and identify functional parameters that are different from or the same as those of the phosphate transport protein. The pyruvate transport system (monocaboxylate transporter, proton compensated, electroneutral) appears to be present in all mitochondria studied even though it may occur with different maximum turnover rates or concentrations in mitochondria from different organs or different organisms. The glutamate transport activity has already been demonstrated to be absent from some types of mitochondria while again different activities in other mitochondria may be due to differences in specific activities or transport protein concentrations. The glutamate transporter also is proton compensated and electroneutral. We plan to identify the transport proteins in dodecyl sulfate polyacrylamide gels after electrophoresis on the basis of labeling with radioactive SH reagents. While it is important to identify the labeled protein, it is necessary that the protein is detected on stained gels (Silver or Coomassie Blue stain) in order to determine its presence in the various fractions during stages of purification. We expect to be able to use our newly modified reconstitution procedure to incorporate the proteins in an active form into liposomes in order to study their properties in detail. Mitochondria are of critical importance to the cellular metabolism and metabolic anions have to be transported into the mitochondrial matrix. The only anion transport system which are not metabolite transporters as such but for which we are beginning to develop an understanding are the adenine nucleotide translocase (electrogenic anion exchange transporter) and the phosphate transporter (proton compensated electroneutral transporter). It is thus of fundamental importance to identify and characterize transport proteins that are responsible for anion metabolite transport.